miR-1306 Mediates the Feedback Regulation of the TGF-ß/SMAD Signaling Pathway in Granulosa Cells

Yang, Liu; Du, Xing; Liu, Lu; Cao, Qiuyu; Pan, Zengxiang; Li, Qifa

We also identified and characterized the core promoter of the porcine miR-1306 gene as well as the binding regions for transcription factors such as breast cancer 1 (BRCA1), Sp1 transcription factor (SP1), and upstream transcription factor 2 (USF2) (Supplementary Figure 7) . We detected four SMAD4-binding elements (SBEs) (Figure 5A ), indicating that SMAD4 may act as a transcription factor and regulate miR-1306 transcription. To confirm this, recombinant reporter vectors containing SBEs (wild-type or mutant-type) were constructed ( Figure 5B ) and co-transfected along with SMAD4 Further, we identified the core promoter of the porcine miR-1306. The putative promoter of the porcine miR-1306 was predicted by using University of California, Santa Cruz (UCSC) database (Supplementary Figure S6A,B) . Luciferase assay indicated that the region from −230 nt to 699 nt is the core promoter of porcine miR-1306 gene ( Figure 4B,C) . Furthermore, we observed that the activity of miR-1306 gene core promoter was dramatically suppressed by SMAD4 overexpression (Figure 4D ), suggesting that miR-1306 gene transcriptional activity is regulated by SMAD4. Taken together with the results of our previous study [9] , we conclude that the transcription factor SMAD4 attenuates miR-1306 expression in porcine GCs by inhibiting its transcriptional activity.